Quantifying Mitochondrial Morphology

Assessment of mitochondrial morphology was carried out as previously described [15 (link)]. Cells were seeded in 6-well plates, trypsinized, and washed with phosphate buffered saline and centrifuged (900 x g, 1 min). 100μl of the cell suspension was centrifuged (450 x g, 4 min; Shandon Cytospin 4, Thermo Fisher Scientific) using cytological funnels together with silane-coated glass slides [29 (link)]. For immunostaining, cells were fixed in 4% paraformaldehyde (1 h, room temperature (RT)) and blocked with 3 % bovine serum albumin. Mitochondria were incubated with anti-Tom20 antibody and appropriate secondary antibody. Confocal images were obtained on Zeiss LM510 inverted microscope (Carl Zeiss, Jena, Germany) with appropriate argon lasers (488nm) and 40X objective. Mitochondrial phenotype of each cell was categorized as tubular or fragmented, as previously described [29 (link)]. A cell with “fragmented mitochondria” was defined as the cell with 8 or more individual mitochondrial fragments that were each 3 μm in length across the longest axis. At least 300 cells were analyzed per experimental group.